Production and properties of phytase and acid phosphatase from a sake koji mold, Aspergillus oryzae.

We identified three types of acid phosphatase (ACP-I, ACP-II, and ACP-III) produced by Aspergillus oryzae in a submerged culture using only phytic acid as the phosphorous substrate. The optimum pH for the activities of the three enzymes was in the range of 4.5 to 5.5. Analysis of the substrate specificities of these enzymes revealed that ACP-I and ACP-III were acid phosphatases, and ACP-II was a phytase. These enzymes were produced during different periods of mycelial growth: ACP-II was produced during the early phase of cultivation (around 24 h), and ACP-I was produced between 24 to 72 h. ACP-III was detected after the production of ACP-I and ACP-II had ceased. The release of phosphate from phytic acid was expected to be due to the cooperative hydrolysis of these enzymes.

Stimulation of porphyrin production by application of an external magnetic field to a photosynthetic bacterium, Rhodobacter sphaeroides.

The effects of an external magnetic field on the production of porphyrin were investigated using Rhodobacter sphaeroides IF012203 under anaerobic-light conditions. Upon application of a 0.13-0.3-T magnetic field, the growth was slightly suppressed and porphyrin extracellular production was activated at both the N and S poles, particularly at the N pole up to about 5.3 times that in the control experiment (6.7 mg/1) (without magnetic filed application). The maximum production was 35.8 mg/1 at the N pole with a magnetic field of 0.3 T. At the same time, the 5-aminolevulinic acid dehydratase (ALAD) concentration was enhanced in the cells at the N and S poles, but particularly at the the N pole. This suggested the possibility that the magnetic field might activate ALAD gene expression in R. sphaeroides IF012203 and result in enhanced porphyrin production.

Production of two types of phytase from Aspergillus oryzae during industrial koji making.

In our previous study, it was determined that phytase produced by Aspergillus oryzae plays an important role in supplying phosphate to yeast in the process of making sake. During koji making, two types of phytase (Phy-I and Phy-II) are produced. The purified phytases have high thermal and pH stability, in comparison to phytase purified from a submerged culture (ACP-II). In the present study, Phy-I and Phy-II retained their activities for 45 h. The NH2-terminal sequence of Phy-1, which is eight amino acids in length, was identical to that of ACP-II, but the molecular weights of these two forms, as estimated by SDS-PAGE, were quite different from each other (Phy-I, 120 kDa; ACP-II, 58 kDa). From the NH2-terminal amino acid sequence analysis of the predominant phytase (Phy-II), a molecular weight of 116 kDa was expected to reflect a new type of phytase produced only in koji culture. The substrate specificity of Phy-II was sufficiently broad that it hydrolyzed not only phytic acid and p-nitro phenyl phosphate, but also glucose 6-phosphate and glycerol 1-phosphate. In the process of making koji, Phy-I was produced at an early stage, followed by Phy-II; with both phytases being thought to function to hydrolyze phytic acid cooperatively.

Production of cellulose- and xylan-degrading enzymes by a koji mold, aspergillus oryzae, and their contribution to the maceration of rice endosperm cell wall.

The production of cellulose- (CEL), xylan- (XYL), and pectin-degrading enzymes (PEC) by a koji mold, Aspergillus oryzae, was studied, and their contributions to the maceration of the rice endosperm cell wall were investigated with regard to the utilization of available rice in the sake mash. The sake koji mold showed higher CEL and XYL productivities, whereas the miso and soy sauce koji molds showed higher PEC productivity. Statistical analyses indicated that CEL and XYL contribute predominantly and synergistically to the maceration of the rice endosperm cell wall. A. oryzae produced at least three kinds of CEL (Cel-1, 2, 3) and two kinds of XYL (Xyl-1, 2) when cultured in a wheat bran medium. In the solid-state culture, the production of Cel-3 and Xyl-2 was markedly stimulated by decreasing the moisture content of the solid substrate, although the production levels of Cel-1 and Xyl-1 were almost the same. These data suggest that the production of Cel-3 and Xyl-2 is strongly influenced by culture conditions, and that water activity is one of the dominant factors in the regulation of their production.

Properties of cellulose-degrading enzymes from Aspergillus oryzae and their contribution to material utilization and alcohol yield in sake mash fermentation.

Four cellulose-degrading enzymes were identified in a solid-state culture of Aspergillus oryzae. The three major enzymes were purified and named Cel-1, Cel-2, and Cel-3, respectively. The molecular weights were determined to be 62, 120, and 34 kDa, respectively. The optimum temperature of Cel-3 activity was higher than that of the other enzymes. An acidic pH was found to be more suitable for Cel-1 activity than for the other enzymes, and Cel-3 was more stable under acidic conditions than the other two. These properties and the results of a protein homology search for N-terminal amino acid sequences suggest that Cel-1 and Cel-3 correspond to the previously isolated endo-1,4-beta-glucanase CelB and CelA, respectively. The analysis of substrate specificity suggested that Cel-2 is likely to be beta-glucosidase. The effect of Cel-1, Cel-2, and Cel-3 on the sake mash fermentation was determined and it was found that Cel-2 markedly improved material utilization and alcohol yield in sake mash fermentation.